Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillatory tangles present in the brain, particularly in those regions of the brain involved in memory and cognition. β-amyloid peptide (Aβ) is a 39-43 amino acid peptide that is major component of amyloid plaques and is produced by cleavage of a large protein known as the amyloid precursor protein (APP) at a specific site(s) within the N-terminal region of the protein. Normal processing of APP involves cleavage of the protein at point 16-17 amino acids C-terminal to the N-terminus of the α-AP region, releasing a secreted ectodomain, α-sAPP, thus precluding production of β-AP. Cleavage by a putative β-secretase enzyme of APP between Met671 and Asp672 and subsequent processing at the C-terminal end of APP produces Aβ peptide which is highly implicated in Alzheimer's pathology (Seubert, et al., in Pharmacological Treatment of Alzheimer's disease, Wiley-Liss, Inc., pp. 345-366, 1997; Zhao, J., et al. J. Biol. Chem. 271: 31407-31411, 1996).
It is not clear whether β-secretase enzyme levels and/or activity are inherently higher in Alzheimer's patients than normal; however, it is clear that the cleavage product of this enzyme, Aβ peptide, is abnormally concentrated in amyloid plaques in the brain of these patients. Therefore, it is desirable to isolate, purify and characterize the enzyme responsible for the pathogenic cleavage of APP in order to help answer this and other questions surrounding the etiology of the disease. In particular, it is also desirable to utilize the isolated enzyme, or active fragments thereof, in methods for screening candidate drugs for ability to inhibit the activity of β-secretase in in vitro systems. Drugs exhibiting inhibitory effects on β-secretase activity are expected to be useful therapeutics in the treatment of Alzheimer's disease and other amyloidogenic disorders characterized by deposition of Aβ peptide containing fibrils.
U.S. Pat. No. 5,744,346 (Chrysler, et al.) describes the initial isolation and partial purification of β-secretase. The present invention provides a significant improvement in the purity of enzyme, by providing a purified β-secretase enzyme that is at least 200 fold purer. Such pure enzyme has utility in a number of applications, including crystallization for structure determination. The invention also provides the methods for producing recombinant forms of human and mouse β-secretase. It is a further discovery of the present invention that human β-secretase is a so-called “aspartyl” (or “aspartic”) protease, which is inhibited by certain polypeptides having 5 to 14 amino acids including polypetpides containing the non-natural amino acid residue, statine, as well as inhibitors containing a hydroxyethylene as an isosteric replacement of a peptide amide bond.
Additionally U.S. Pat. No. 5,192,668 entitled “Synthesis of Protease Inhibitor,” issued Mar. 9, 1993; U.S. Pat. No. 5,188,950 entitled “Method of Preparing HIV Protease Inhibitors,” issued Feb. 23, 1993; U.S. Pat. No. 5,187,074 entitled “Method of Hydroxylation with ATCC 55086,” issued Feb. 16, 1993; and U.S. Pat. No. 5,175,298 entitled “Dipeptide Hydroxy Ethylene Isostere Synthesis and Intermediate Therefor,” issued Dec. 29, 1992, relate to the synthesis and use of hydroxyethylene isosteres as protease inhibitors and are hereby incorporated in their entirety with the same effect as if each were incorporated separately by reference.